This invention relates to the therapeutic induction of apoptosis in inflammatory cells by introducing into those cells a gene which induces apoptosis (programmed cell death or non-necrotic cell death) in these cells. The Apoptosis-Inducing Gene (which will sometimes be referred to herein as AIG) is driven by a TNFxcex1 promoter (TNFp) or other inducible gene activated in inflammation. In one embodiment, apoptosis is selectively induced in those cells capable of producing TNFxcex1. The TNFp-AIG or other chimeric gene may be conveniently introduced in vivo using conventional gene therapy techniques. Advantageously, in the embodiment wherein the chimeric gene is TNFp-AIG, it is expressed in only those cells producing the inflammatory cytokine, TNFxcex1. In addition, since the TNFp-AIG chimeric gene contains the TNFxcex1 promoter elements, it also sequesters inducible, TNFp-selective transcription factors. Such sequestration results in a reduction in endogenous production of TNFxcex1. The present invention relates specifically to TNFp-AIG and similar gene constructs, cells containing chimeric genes, methods for induction of apoptosis in cells transfected with chimeric genes, pharmaceutical compositions containing chimeric genes, methods for in vitro selection of TNFxcex1 non-producer somatic cell variants within a TNFxcex1 producing cell population and the like, a method for identifying dominant negative/dominant suppressive genes responsible for inhibiting TNFxcex1 production and therapeutic methods using the chimeric gene.
In many inflammatory conditions, cytokines such as IL-1, IL-10, GM-CSF and TNFxcex1 are excessively produced as a result of mass aggregation and accumulation of inflammatory cells (Brennan F. M. et al., British Medical Bulletin 1995, 51/2, 368-384). Upregulation and/or disregulation of cytokines in inflamed tissue may be directly or indirectly responsible for exacerbation of chronic inflammatory diseases. For example, the most marked pathology in rheumatoid arthritis (RA) is displayed at the local site of inflammation (i.e., the synovial joints). Therefore, it is likely that the cytokines produced in the synovial joints of RA patients play an important role in the disease process. Of those cytokines, IL-1 and TNFxcex1 are believed to be responsible for the devastating cartilage destruction and bone erosion which characterizes RA (Dayer J. M. et al., J. Exp. Med., 1985, 162, 1208-1215; Gowen M. et al., Nature, 1983, 306, 378-380). The presence of excessive amounts of IL-1 and TNFxcex1 in the synovial joints has been shown to accelerate development of collagen-induced arthritis in rodents (Brennan F. M., et al., Clin. Expt. Immunol., 1994, 97/1, 1-3). Excessive amounts of TNFxcex1 and IL-1 are produced in the synovial tissue by a variety of cell types at the cartilage-pannus junction, including cells of the macrophage lineage, macrophage-like synoviocytes, activated T-cells and possibly fibroblast-like synoviocytes (Chu C. Q. et al., Arthritis and Rheumatism, 1991, 34, 1125-1132; Deleuran B. W., et al., Arthritis and Rheumatism, 1992, 35, 1170-1178).
In addition to the above described inflammatory effects, TNFxcex1 plays a ubiquitous and key role in a variety of pro-inflammatory events, such as induction of IL-1 activity in monocytes. Indeed, anti-TNFxcex1 neutralizing antibodies have been shown to reduce overall IL-1 production (Portillo, et al., Immunol., 1989, 66, 170-175; Brennan F. M., et al., British Medical Bulletin 1995, 51/2, 368-384). Thus, an added benefit to blocking the effect of the inflammatory cytokine TNFxcex1 is the reduction in production of the equally destructive pro-inflammatory mediator, IL-1. Furthermore, it is well known that TNFxcex1 is a transcriptional activator of other inflammation-related genes. For example, the presence of TNFxcex1 stimulates production of other cytokines (such as GM-CSF) and cell surface receptors, including HLA class II antigens and adhesion molecules (Alvaro-Garcia J. M., et al., J. Exp. Med., 1989, 146, 865-875), which, results in continuous recruitment of activated T cells and neutrophils resulting in synovial inflammation and hyperplasia and ultimately, in augmented destruction of cartilage and bone (Allen J. B., J. Exp. Med., 1990, 171, 231).
Conventional therapy against inflammatory disorders is typically directed against symptomatic inflammation. Such therapies provide only temporary relief without significantly delaying disease progression. In contrast, therapies targeting TNFxcex1 and other factors induced in the inflammatory process are likely to be more promising. For example, in collagen-induced arthritis animal models, an anti-TNFxcex1 antibody and soluble TNFxcex1 receptor-IgG chimera effectively reduced paw swelling, joint involvement and cartilage and bone destruction (Williams R. O. et al., Proc. Natl. Acad. Sci., 1992, 89, 9784-9788). Human trials using both humanized anti-TNFxcex1 antibodies and TNFxcex1 receptor-IgG chimeric molecules produced dramatic results (Elliott M. J., et al., Arthritis and Rheumatism, 1993, 36, 1681-1690; Elliott M. J., et al., Lancet, 343, 1105-1110). Although treatment with these TNFxcex1 antagonists appears to be well tolerated, it also results in production of antibodies against the recombinant proteins. Thus, these therapies may not be suitable for long term treatment and do not achieve true disease abatement. In order to actually modify progression of the disease, TNFxcex1 must be continuously targeted using TNFxcex1-specific therapies. Such a therapeutic protocol is impractical with these biologic agents and would be difficult to administer in the long term.
In an alternate therapeutic option, inflamed synovium may be removed using surgical (Herold N. and Schroder H. A., Acta Orthop. Scand., 1995, 66, 252-254; Ogilvie-Harris D. J. and Weisleder L., Arthroscopy, 1995, 11, 91-95), chemical (Cruz-Esteban C. and Wilke W. S., Bailliere""s Clinical Rheumatol., 1995, 9, 787-801) or radiation-induced synovectomy (Cruz-Esteban C. and Wilke W. S., Bailliere""s Clinical Rheumatol., 1995, 9, 787-801). The results following arthroscopic surgical synovectomy are good, showing improvement from the preoperative condition to the postoperative condition. Non-surgical synovectomy is performed using various chemical agents such as osmic acid, alkylating agents such as nitrogen mustard and thiotepa, methotrexate. Unfortunately, non-surgical synovectomies (including chemical and radiation-induced) are procedurally complicated, provide only short term relief and show only patchy reduction of the synovial hyperplasia. Furthermore, most of the non-surgical alternatives are potential teratogens. In addition, the chemical damage to afflicted tissue in non-surgical synovectomy, as well as surgically-induced tissue damage, often cause an inflammatory response themselves. Finally, it should be noted that these approaches suffer from the risks and side-effects commonly associated with conventional pharmaceutical therapy and invasive surgical procedures, including the expense and inconvenience of hospitalization and rehabilitation.
Accordingly, a need still exists for an effective therapeutic approach to treating inflammatory disorders in general and RA in particular.
This invention overcomes the drawbacks associated with previous therapies for treating inflammatory disorders by providing a novel therapeutic approach. According to one embodiment of this invention, apoptosis is selectively induced in TNFxcex1-producing inflammatory cells, causing destruction of these cells without an associated inflammatory reaction.
One objective of this invention is to provide a therapeutic method comprising the step of introducing into the inflammatory cells of a mammal, or cells at a site of inflammation, a chimeric gene containing a self-regulating apoptosis-inducing gene (AIG). The AIG is driven by a promoter such as a TNFxcex1 promoter (TNFp; see FIGS. 1 and 2), and, preferably, a promoter enhancer. Therefore, it is expressed in all and only those cells capable of producing TNFxcex1.
Another objective of this invention is to provide TNFp-AIG and the like chimeric gene constructs, processes for making them, methods of using them, and preparations containing them.
Yet a further objective of this invention is to provide a method for the induction of apoptosis in cells transfected with the TNFp-AIG chimeric gene, a method for the in vitro selection of TNFxcex1 non-producer somatic cell variants in a population, a method for identifying dominant/negative genes responsible for the genesis of a TNFxcex1 non-producing population and a method for identifying products responsible for regulation of TNFxcex1 production (FIG. 10).
These and other objectives will be readily appreciated by those of ordinary skill in the art based upon the following detailed disclosure of the invention.